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A delaTorre

Publications and source records attributed to A delaTorre.

6 recordsLinked to original sources
PubMed1999

Endotoxin-mediated S-nitrosylation of p50 alters NF-kappa B-dependent gene transcription in ANA-1 murine macrophages.

Nitric oxide (NO) regulates cellular function, in part, by S-nitrosylating active site thiol groups of proteins. Ex vivo S-nitrosylation of NF-kappa B p50 significantly decreases its capacity for DNA binding. To determine the cellular relevance of this observation, we utilized the ANA-l murine macrophage model of endotoxin (LPS)-mediated NO synthesis. In selected instances, the NO synthase inhibitor, L-arginine methyl ester (L-NAME; 100 microM), or the NO donor, S-nitroso-N-acetylcysteine (SNAC; 100 microM), was added. In contrast to that of LPS cells, nuclear extracts from LPS + L-NAME cells demonstrated increased NF-kappa B DNA binding on gel shift analysis. Addition of SNAC to LPS + L-NAME cells restored binding to a level equivalent to that of LPS cells. Spectrophotometric analysis of NF-kappa B p50 immunoprecipitates demonstrated S-NO bonds exclusively in LPS cells; these p50 protein isolates retained the same DNA binding characteristics as that of the nuclear extracts. Transfection assays utilizing NF-kappa B-dependent promoter-reporter constructs demonstrated increased activity in LPS + L-NAME cells compared with LPS cells; nuclear run-on assays confirmed increased transcription of the corresponding genes. These results suggest that LPS-mediated NO synthesis is associated with S-nitrosylation of NF-kappa B p50 and inhibition of NF-kappa B-dependent DNA binding, promoter activity, and gene transcription. We conclude that NO can regulate gene transcription by S-nitrosylation of NF-kappa B.

Animals↗
PubMed1998

Differential effects of nitric oxide-mediated S-nitrosylation on p50 and c-jun DNA binding.

BACKGROUND: Nitric oxide (NO) regulates a variety of cellular functions. One mechanism by which NO may exert its influence is through formation of S-nitrosothiols at critical thiol residues in protein-active sites, including those of nuclear protein transcription factors. METHODS: NF-kappa B p50 and AP-1 c-jun were S-nitrosylated in the presence of acidic NaNO2. Wild-type protein and protein subjected to nitrosylating conditions in the absence of NaNO2 served as controls. Confirmatory evidence for S-nitrosothiol bond formation was obtained by ultraviolet-visible spectrophotometry with the absorption maximum for S-NO bonds at approximately 320 to 360 nm. With consensus oligonucleotide probes, gel-shift analysis was used to examine DNA binding characteristics. RESULTS: In the case of NF-kappa B p50, S-nitrosylation resulted in significantly decreased DNA binding. In contrast, S-nitrosylation did not alter c-jun DNA binding. The S-nitrosylating conditions themselves did not alter p50 or c-jun DNA binding. Quantitative analysis was performed according to the Scatchard plot technique to generate the respective dissociation constants. S-nitrosylated p50 was associated with a fourfold greater dissociation constant than that of the wild-type p50. CONCLUSIONS: S-nitrosylation of transcription factors may be one mechanism by which NO may selectively regulate gene transcription.

Carcinogens↗
PubMed1997

CD14-dependent mechanism for endotoxin-mediated nitric oxide synthesis in murine macrophages.

Endotoxin-mediated macrophage synthesis of nitric oxide (NO) is associated with immune effector function, intercellular communication, leukocyte adhesion, vascular integrity, and neurotransmission. However, little is known of the cellular receptor and signal transduction pathway by which endotoxin induces NO production. With the use of a model of ANA-1 murine macrophages, we stimulated NO production by incubation with increasing concentrations of endotoxin and 5% fetal calf serum. In selected instances, the anti-CD14 antibody, ED9, was added. Endotoxin-mediated NO synthesis was dependent on CD14 function and the presence of an additional serum factor. Endotoxin treatment increased plasma membrane GTPase activity and 35S-labeled guanosine 5'-O-(3-thiotriphosphate) ([35S]GTP gamma S) binding. Conversely, coincubation of cells with endotoxin and the heterotrimeric G protein inhibitors, suramin and guanosine 5'-O-(2-thiodiphosphate) trilithium salt, was associated with decreased NO synthesis, plasma membrane GTPase activity, and [35S]GTP gamma S binding. Blockade of CD14 or G protein function was associated with ablation of endotoxin-mediated inducible NO synthase (iNOS) protein expression, iNOS mRNA levels, and iNOS gene transcription, as determined by immunoblot, reverse transcriptase-polymerase chain reaction, and nuclear run-on analyses, respectively. These results indicate that endotoxin-mediated NO synthesis is a CD14-heterotrimeric G protein-dependent process.

Analysis of Variance↗